In contrast to universally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins will drastically reduce the range of relative protein abundances within complex tissue proteomes, and greatly enhance the proteome coverage using the CITP-based proteomic technology. Our proposed research efforts therefore aim to fully characterize, develop, and exploit the use of this differential concentration effect to achieve comprehensive proteome analysis of clinical specimens with limited sample availability. The laser capture microdissection (LCM) process provides a rapid and straightforward method for isolating selected subpopulations of cells for downstream biochemical and molecular analyses. On the basis of cell enrichment, LCM also serves as a targeted sample fractionation approach toward the reduction in protein complexity and relative abundance. The proposed coupling of tissue microdissection for diseased cell enrichment with CITP-based selective analyte concentration not only presents a synergistic strategy for the detection and characterization of low abundance proteins, but also offers a novel biomarker discovery paradigm for enabling the identification of tumor-associated markers, exploration of molecular relationships among different tumor states and phenotypes, and a deeper understanding of molecular mechanisms that drive cancer progression. PUBLIC HEALTH RELEVANCE: By combining our unique bioanalytical capabilities with the expertise of Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS) in tissue microdissection and neuropathology, the proposed research represents a synergistic effort toward the development, evaluation and validation of a novel biomarker discovery paradigm for enabling the proteomic analysis of tumor cells and their micro-environment in support of cancer research, diagnosis, and treatment.